Gas-liquid chromatography of carbohydrate derivatives: the separation and quantitative determination of synthetic and naturally occurring glycosyl derivatives of amino acids

A gas-liquid chromatographic separation of the trimethylsilyl derivatives of several synthetic and naturally occurring glycosyl derivatives of amino acids is described. This technique may be used for the qualitative and quantitative determination of several compounds of biological interest including 3-O-[beta]--xylopyranosyl--serine, 3-O-[beta]--arabinopyranosyl--serine, 5-O-[beta]--galactopyranosyloxy-D,-lysine, and 2-acetamido-N-(-aspart-4-oyl)-2-deoxy-[beta]--glucopyranosylamine. In addition, the mobilities of these glycosyl derivatives of amino acids in paper chromatographic and paper electrophoretic systems are reported.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34111/1/0000395.pd

Biosynthesis of keratan sulfate: Purification and properties of a galactosyltransferase from bovine cornea

A soluble galactosyltransferase was purified 22,000-fold from bovine cornea. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-acetyl--glucosamine, [alpha]- and [beta]-glucosaminides, bovine cornea and nasal septum agalactokeratan, and to glycoproteins containing terminal nonreducing N-acetylglucosaminyl units. When N-acetyl--glucosamine served as acceptor, the product formed by the cornea transferase contained galactose glycosidically linked to carbon atom 4 of N-acetyl--glucosamine; the same glycosidic linkage was found in [14C]keratan preparations isolated from reaction mixtures where keratan containing terminal nonreducing N-acetylglucosaminyl units served as acceptor. The cornea enzyme exhibited a markedly lower Km with keratan than with N-acetyl--glucosamine. The physical and kinetic properties of the cornea galactosyltransferase and of the milk A-protein (A-protein + [alpha]-lactalbumin = lactose synthase), including modulations of acceptor specificity by [alpha]-lactalbumin, were compared. The results of these studies strongly suggest that the two glycosyltransferases are similar, if not identical. Efforts to demonstrate the presence of other soluble galactosyltransferases in cornea were unsuccessful; no change in the ratios of products formed with several acceptors was observed at any stage of purification. It is suggested that in bovine tissues a single galactosyltransferase participates in the synthesis of both high and low molecular weight galactosides including the assembly of the repeating disaccharide [O-[beta]-galactopyranosyl-(1 --> 4)-N-acetylglucosamine] of cornea keratan sulfate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23647/1/0000611.pd

Bovine testicular [beta]-galactosidase: Purification of enzyme fractions that exhibit high affinity for phosphomannosyl receptors

An improved method is described for the preparation of bovine testicular [beta]-galactosidase that allows the isolation of enzyme fractions that bind avidly to phosphomannosyl receptors. The procedure permits removal of a contaminating [beta]-hexosaminidase and yields nearly homogeneous [beta]-galactosidase. Enzyme eluted from DEAE-Sephacel was arbitrarily divided into pools that exhibited differing ability to bind phosphomannosyl receptors. A high binding fraction was rapidly assimilated by cultured cells and bound to both low and high molecular weight phosphomannosyl receptors. Carbohydrate analysis of the high binding fraction indicates an average content of one complex and one high mannose oligosaccharide chain per molecule and an average mannose 6-phosphate content of two residues per molecule. However, electrofocusing studies indicated that all the fractions were heterogeneous with respect to sialic acid and phosphate content. The purification procedure also provides highly purified [beta]-galactosidase suitable for removing [beta]-galactosidase residues from a variety of complex carbohydrates.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27707/1/0000093.pd

The role of glycosidically bound mannose in the assimilation of [beta]-galactosidase by generalized gangliosidosis fibroblasts

Bovine testicular [beta]-galactosidase is rapidly assimilated by generalized gangliosidosis skin fibroblasts. The enzyme contains equimolar amounts of mannose and glucosamine and strongly binds to concanavalin A-Sepharose. Pretreatment of [beta]-galactosidase with a mannosidase preparation from reduced the rate of assimilation of the enzyme 97%. These data indicate that mannosyl residues play a role in assimilation of the enzyme. This conclusion is supported by observed inhibition of [beta]-galactosidase assimilation by mannose, methyl [alpha]- and [beta]-mannopyranosides, and mannose-containing testicular glycoproteins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21625/1/0000004.pd

The interaction of wheat germ agglutinin with keratan from cornea and nasal cartilage

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21827/1/0000230.pd

INTERMEDIARY METABOLISM OF THE SIALIC ACIDS

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75332/1/j.1749-6632.1963.tb16639.x.pd

Metabolism of ovomucoid by the developing chick embryo This work was supported by Grant AM-10531 from the National Institute of Arthritis, Metabolic and Digestive Diseases, National Institutes of Health.

A glycoprotein resembling ovomucoid was purified from chorioallantoic membranes of 12.5 day chick embryos by a sequential procedure involving zinc chloride precipitation, hydroxylapatite chromatography, and ion exchange chromatography on DEAE- and CM-cellulose. The resulting preparation was homogeneous as indicated by polyacrylamide gel electrophoresis at 3 pH values, isoelectric focusing, ultracentrifugation, and immunochemical analysis. The purified material had an isoelectric point of 4.8, molecular weight of 29,500 and contained 1.6 moles of galactose, 7.7 moles of mannose, 22.8 moles of N -acetylglucosamine and 0.4 mole of glucose per mole of protein. The ovomucoid fraction inhibited bovine trypsin, forming a 1 to 1 complex on a molar basis, and elicited a positive anaphylactoid response in albino rats at levels as low as 0.5 Μg. A specific ovomucoid antiserum was obtained from rabbits that allowed investigation of the mobilization and metabolism of ovomucoid in the developing chick embryo. These studies revealed that between days 12 and 13 of embryo development a progressive migration of ovomucoid occurs from the albumin sac to the amnionic sac. The glycoprotein is then swallowed and transported through the gut to the yolk sac, where it is catabolized by proteases and glycosidases contained in the yolk sac membrane. Between day 12 of embryo development and day 7 after hatching, ovomucoid appeared in the serum of the chick and reached a maximum value of 2.4 mg % on day 18 of embryo development.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38074/1/1401890203_ftp.pd

Automated assay of glycosidases

The Technicon Basic AutoAnalyzer sampler system was modified for simultaneous sampling of glycosidase(s) and substrate-buffer solutions. The inexpensive modification allows performance of automated enzyme analyses and enzyme kinetic studies with minimal consumption of substrate and/or enzyme.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22498/1/0000041.pd

The enzymatic synthesis of sialyl-lactose

Sialyl-lactose (SL) was first isolated from lactating rat mammary gland by Trucco and Caputto (1954). In subsequent extensive studies on milk oligosaccharides, Kuhn and coworkers ( Kuhn and Brossmer, 1958 and Kuhn, 1959) obtained two isomers of SL from human milk, one being identical to SL from bovine colostrum. Bovine SL was shown to be N-acetylneuraminyl-(2 --> 3)-[beta]-D-galactopyranosyl-(1 --> 4)-D-glucose, i.e., SL-(2 --> 3). The other SL from human milk is N-acetylneuraminyl-(2 --> 6)-[beta]-D-galactopyranosyl-(1 --> 4)-D-glucose, i.e., SL-(2 --> 6).We now wish to report the enzymatic synthesis of sialyl-lactose by a particulate preparation from rat mammary gland. Chromatographic and periodate oxidation data indicate that the product is SL-(2 --> 3). In addition, preliminary evidence shows that this preparation catalyzes the synthesis of other trisaccharides, such as sialyl-galactosyl-acetylglucosamine. .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32234/1/0000295.pd